Energy-filtered electron microscopy and electron energy loss spectroscopy (EELS) have been used to characterize the distributions of sulfur-rich proteins in stratifying epithelia and their redistribution in the cornified layer, with particular attention to assembly of the cornified cell envelope (CE). Measurements provided quantitative information that complements highly specific but qualitative data from immunocytochemistry. Spatial distributions of sulfur, phosphorus and nitrogen were mapped in unstained thin sections of mouse epidermis and forestomach, using electron spectroscopic imaging. Nitrogen distributions were indicative of total protein while phosphorus images provided a control to validate the algorithms used to calculate the elemental maps. Sulfur was found at high levels in round L-granules in the granulocyte cytoplasm and in the cornified CE, correlating with the presence of the protein, loricrin (~ 7% Cys/Met residues). EELS measurements in the scanning transmission electron microscope confirmed these observations quantitatively. These data also confirm that approximately 75% of the protein in the CE is loricrin, as inferred from modeling of its amino-acid composition. Extracellular deposits were also observed between the cornified squames in fetal mouse epidermis. The sulfur content of these peripheral deposits is at least as high as that of L-granules but they do not label with anti-loricrin antibodies, suggesting the presence of another sulfur-rich protein. This is a continuation of Intramural Research Project Z01-RR-10481-02 BEI.